PROJECT SUMMARY Circulating insulin-like growth factor (IGF)-I, critical for all phases of human development, growth, and maturation, is produced predominantly by the liver, with post-natal hepatic production of IGF-I highly dependent on pituitary-secreted growth hormone (GH). The importance of the hepatic human GH-IGF-I axis for normal human growth is highlighted by the multiple genetic defects downstream of GH, in children who present with severe growth failure (height -3 to -10 standard deviation below the mean), GH insensitivity (GHI) and IGF-I deficiency (IGFD). The identification of rare mutations of STAT5B (signal transducer and activator of transcription 5B) in children with GHI and IGFD, together with the more than 80 reported GHR (GH receptor) mutations in affected children, firmly established the critical importance of the GHR-STAT5B pathway for GH- induced regulation of IGF-I production. However, a disturbing >60% of GHI, idiopathic short statured children, do not carry GHR or STAT5B defects, yet are IGF-I deficient, indicating still limited understanding of the hepatic GHR-STAT5B-IGF-I growth axis and that current model systems are clearly insufficient. We propose establishing a new patient-based model for deliverable assessment of GHI and IGFD, based on emerging human induced-pluripotent stem cells (iPSC)-derived organoids (?mini-organs?), which are promising ?living replica? models for disease investigation and precision medicine. We hypothesize that human hepatic response to GH cues can be recapitulated in patient iPSC-derived liver organoids. This human- based model offers unique opportunities to probe human hepatic GHR-STAT5B dependent and independent mechanisms of gene regulation in patient-specific organoids and to follow responsiveness of the developing liver organoid to hormonal cues, paralleling in utero and post-natal developmental processes. The Specific Aims will leverage our unique panel of characterized, patient primary cells to assess molecular signatures, sensitivity, and functionality of iPSC-derived organoids to GH cues, towards establishing the first viable human based models for mechanistic investigation of the human hepatic GH-IGF-I growth axis. The Specific Aims will (1) assess the developmental expression of the GHR-STAT5B-IGF-I axis in human iPSC-derived liver organoid model; and (2) delineate hepatic STAT5B-dependent and independent GH-regulated gene expression in human iPSC-derived liver organoids. At the conclusion of these studies we will have established and demonstrated the utility of a patient-specific, iPSC-derived liver organoid system for evaluating functional disruptions by genetic defects leading to GHI and IGFD. New mechanistic insights gained include improved understanding of human, hepatic GHR-STAT5B mediated regulation of IGF-I expression, with potential for identifying new markers of human GH responsiveness. In the long term, these novel patient-based models provide a platform for future personalized therapeutic testing, towards improving patient management and care of affected children, and for future evaluation of liver responsiveness to other hormonal cues.